Dear Bernhard Our campaigns over the years aiming to produce mammalian cytokines and the ectodomains of cytokine receptors via eukaryotic expression systems (mainly in several HEK293 flavors) for structural biology, have taught us that the N-linked glycosylation issue remains a very empirical exercise. Our protein targets are typically 15-70 kDa and have 2-6 predicted N-linked glycosylation sites. For instance, we have seen that elimination of even one such site by mutagenesis can abrogate protein secretion. So for those cases one may even make an argument for a glycosylation ‘hotspot'. Also, eliminating all possible N-linked glycosylation sites at once in the couple of cases tried has been synonymous with zero protein secretion. Our consensus of the ‘magic combo’ in terms of expression levels and stability is a reduction of N-linked glycosylation sites by up to 1/3. Such reduced levels of glycosylation also appear to be amenable for enzymatic glycan trimming in subsequent stages of sample preparation with good results. The article http://dx.doi.org/10.1016/j.sbi.2013.04.003 by Aricescu and Owens may provide some additional perspectives. best wishes Savvas
> On 11 Apr 2017, at 22:34, Bernhard Rupp <hofkristall...@gmail.com> wrote: > > Hi Fellows, > > a humble question for our glyco-expressionists: > > I have mutated out the Asns of the N-glycoslation consensus sites for Asp > (Asp simply because the PNGaseF treated protein stays stable so I thought > that might be a good guess) > and indeed the unglycosilated mutant expresses well and gets secreted as > planned. > > But rumor has it that glycoproteins that are mutated to non-glyc often are > not processed correctly and > that we had just dumb luck. > > May I poll the educated opinion of the erudite here? > > Cheers, BR > > ------------------------------------------------------ > Bernhard Rupp > Crystallographiae Vindicis Militum Ordo > http://www.hofkristallamt.org/ > b...@hofkristallamt.org > +1 925 209 7429 > +43 767 571 0536 > ------------------------------------------------------ > :(){ :|: & };: > ------------------------------------------------------
